The present invention relates to the use of enzymes which are associated with the cell (ectoenzymes) and secreted enzymes for monitoring cellular proliferation.
Reporter enzymes are enzymes whose activities are easily assayed when present inside cells. In order to study the regulation of a gene whose expression is regulated by various environmental and/or cellular factors or influences, a gene encoding a reporter enzyme may be fused to the coding region or to the regulatory region of the regulated gene. Reporter genes may be used to determine whether a sequence contains a promoter or other cis-acting element which directs transcription, such as an enhancer. In addition, reporter genes may be used to identify regulatory sites in promoters or other cis-acting elements and to determine the effects of mutating these regulatory sites on the level of gene expression directed by the promoters or other cis-acting elements. Reporter genes may also be used to detect successful transformation, to monitor gene expression under various conditions, to assess the subcellular location of an expressed protein and to identify drugs such as antibiotics.
Since the discovery of penicillin, the use of antibiotics to treat the ravages of bacterial infections has saved millions of lives. With the advent of these xe2x80x9cmiracle drugs,xe2x80x9d for a time it was popularly believed that humanity might, once and for all, be saved from the scourge of bacterial infections. In fact, during the 1980s and early 1990s, many large pharmaceutical companies cut back or eliminated antibiotics research and development. They believed that infectious disease caused by bacteria finally had been conquered and that markets for new drugs were limited. Unfortunately, this belief was overly optimistic.
The tide is beginning to turn in favor of the bacteria as reports of drug resistant bacteria become more frequent. The United States Centers for Disease Control announced that one of the most powerful known antibiotics, vancomycin, was unable to treat an infection of the common Staphylococcus aureus (staph). This organism is commonly found in our environment and is responsible for many nosocomial infections. The import of this announcement becomes clear when one considers that vancomycin was used for years to treat infections caused by Staphylococcus species as well as other stubborn strains of bacteria. In short, bacteria are becoming resistant to our most powerful antibiotics. If this trend continues, it is conceivable that we will return to a time when what are presently considered minor bacterial infections are fatal diseases.
Over-prescription and improper prescription habits by some physicians have caused an indiscriminate increase in the availability of antibiotics to the public. The patients are also partly responsible, since they will often improperly use the drug, thereby generating yet another population of bacteria that is resistant, in whole or in part, to traditional antibiotics.
The bacterial pathogens that have haunted humanity remain, in spite of the development of modern scientific practices to deal with the diseases that they cause. Drug resistant bacteria are now an increasing threat to the health of humanity. A new generation of antibiotics is needed to once again deal with the pending health threat that bacteria present.
As more and more bacterial strains become resistant to the panel of available antibiotics, new antibiotics are required to treat infections. In the past, practitioners of pharmacology would have to rely upon traditional methods of drug discovery to generate novel, safe and efficacious compounds for the treatment of disease. Traditional drug discovery methods involve blindly testing potential drug candidate-molecules, often selected at random, in the hope that one might prove to be an effective treatment for some disease. The process is painstaking and laborious, with no guarantee of success. Today, the average cost to discover and develop a new drug exceeds US $500 million, and the average time from laboratory to patient is 15 years. Improving this process, even incrementally, would represent a huge advance in the generation of novel antimicrobial agents.
Newly emerging practices in drug discovery utilize a number of biochemical techniques to provide for directed approaches to creating new drugs, rather than discovering them at random. For example, gene sequences and proteins encoded thereby that are required for the proliferation of a microorganism make excellent targets since exposure of bacteria to compounds active against these targets would result in the inactivation of the microorganism. Once a target is identified, biochemical analysis of that target can be used to discover or to design molecules that interact with and alter the functions of the target. Use of physical and computational techniques to analyze structural and biochemical properties of targets in order to derive compounds that interact with such targets is called rational drug design and offers great potential. Thus, emerging drug discovery practices use molecular modeling techniques, combinatorial chemistry approaches, and other means to produce and screen and/or design large numbers of candidate compounds.
Nevertheless, while this approach to drug discovery is clearly the way of the future, problems remain. For example, the initial step of identifying molecular targets for investigation can be an extremely time consuming task. It may also be difficult to design molecules that interact with the target by using computer modeling techniques. Furthermore, in cases where the function of the target is not known or is poorly understood, it may be difficult to design assays to detect molecules that interact with and alter the functions of the target. To improve the rate of novel drug discovery and development, methods of identifying important molecular targets in pathogenic microorganisms and methods for identifying molecules that interact with and alter the functions of such molecular targets are urgently required.
To facilitate the identification of new drugs, automated assays which allow the effects of a large number of candidate compounds on microbial proliferation to be easily, rapidly and inexpensively evaluated are required. The present invention relates to the use of ectoenzymes and secreted enzymes in assays for measuring cellular proliferation.
One embodiment of the present invention is a method for measuring cellular proliferation in a sample comprising obtaining a sample of cells which express an ectoenzyme or a secreted enzyme, determining the level of activity of the ectoenzyme or secreted enzyme in the sample and correlating the level of activity of the ectoenzyme or secreted enzyme with the extent of cellular proliferation. The step of determining the level of activity of the ectoenzyme or secreted enzyme may comprise contacting the cells with an agent which yields a detectable product when acted upon by the ectoenzyme or secreted enzyme and determining the level of the detectable product in the sample. The ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) catarrhalis BRO beta-lactamase, P. aeruginosa FpvA ferric pyoverdin receptor, E. coli OmpP endopeptidase, outer membrane phospholipase A, Bacteriodes thetaiotamicron susG starch utilization protein, Haemophilus influenzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N-acetylglucosaminidase, S. pneumoniae neuraminidase, Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. The determining step may comprise determining the level of activity of a secreted enzyme by contacting the growth medium of the cells with an agent which yields a detectable product when acted upon by the secreted enzyme and determining the level of the detectable product in the sample. The determining step may comprise determining the level of activity of a secreted enzyme by contacting a supernatant with an agent which yields a detectable product when acted upon by the secreted enzyme and determining the level of the detectable product in the sample, wherein the supernatant comprises growth media from which the cells have been removed. The ectoenzyme or secreted enzyme may comprise a membrane-bound form of chitobiase. The method may further comprise introducing a gene encoding the membrane-bound form of chitobiase into the cells prior to obtaining the sample of cells. The method may further comprise contacting the cells with sarkosyl. The method may further comprise contacting the cells with sarkosyl and NaCl. The method may further comprise contacting the cells with NaCl. In some versions of the method, the cells are intact. The ectoenzyme or secreted enzyme may be expressed transiently. The ectoenzyme or secreted enzyme may be expressed stably. The ectoenzyme or secreted enzyme may be expressed from a plasmid. The ectoenzyme or secreted enzyme may be endogenous. The ectoenzyme or secreted enzyme may be exogenous. The ectoenzyme or secreted enzyme may be expressed from an inducible promoter. The determining step may comprise determining the level of activity of an ectoenzyme. The method may further comprise preparing a membrane fraction comprising the ectoenzyme. The ectoenzyme or secreted enzyme may be expressed from a gene encoding the ectoenzyme or secreted enzyme which has been introduced into the genomes of the cells. The cells may be selected from the group consisting of prokaryotic cells and eukaryotic cells. The step of determining the level of activity of the ectoenzyme or secreted enzyme may be selected from the group consisting of measuring the amount of a chemiluminescent product produced from a substrate, measuring the amount of a fluorescent product produced from a substrate, measuring the amount of light absorbed by a product produced from a substrate and measuring a decrease in the amount of a detectable substrate. The product maybe p-nitrophenol.
Another embodiment of the present invention is a method for determining the level of membrane-bound chitobiase gene activity in intact cells, comprising the steps of introducing a nucleic acid encoding the membrane-bound chitobiase into a cell population and contacting the cells with a chitobiase substrate.
Another embodiment of the present invention is a gene construct comprising a heterologous promoter operably linked to a nucleic acid encoding a membrane-bound form of chitobiase. The portion of the nucleic acid encoding a membrane-bound form of chitobiase comprises a signal sequence from a gene other than the chitobiase gene.
Another embodiment of the present invention is a cell into which a gene encoding a membrane-bound form of chitobiase has been introduced. The portion of the nucleic acid encoding the membrane-bound chitobiase signal sequence may be heterologous. The gene encoding membrane-bound chitobiase may be introduced into the genome of the cell.
Another embodiment of the present is a method for characterizing a promoter comprising providing a construct comprising the promoter operably linked to a nucleic acid encoding a membrane-bound form of chitobiase, introducing the construct into host cells, and identifying sequences in the promoter which regulate transcription levels. The nucleic acid encoding a membrane-bound form of chitobiase encodes a membrane-bound form of chitobiase may be obtained from an organism selected from the group consisting of Alteromonas sp. 0-7, Arabidopsis thaliana, Bacillus subtilis, Bombyx mori, Bos taurus, Caenorhabditis elegans, Candida albicans, Dictyostelium discoideum, Entamoeba histolytica, Felis catus, Homo sapiens, Korat cats, Lactobacillus casei, Leishmania donovani, Mus musculus, Pisum sativum, Porphyromonas gingivalis, Pseudoalteromonas sp. S9, Rattus norvegicus, Serratia marcescens, Streptomyces plicatus, Streptomyces thermoviolaceus, Sus scrofa, Trichoderma harzianum, Vibrio furnissii, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vulnificus. 
The method of identifying sequences which are involved in regulating transcription may comprise mutagenizing the promoter. The method of identifying sequences which are involved in transcription may comprise constructing deletions in the promoter.
Another embodiment of the present invention is a method for identifying a regulatory element capable of modulating transcription within a test nucleic acid sequence, comprising providing a construct comprising the test nucleic acid sequence operably linked to a nucleic acid encoding a membrane-bound form of chitobiase; introducing the construct into host cells and determining the level of chitobiase activity. The nucleic acid encoding a membrane-bound form of chitobiase may encode a membrane-bound form of chitobiase obtained from an organism selected from the group consisting of Alteromonas sp. 0-7, Arabidopsis thaliana, Bacillus subtilis, Bombyx mori, Bos taurus, Caenorhabditis elegans, Candida albicans, Dictyostelium discoideum, Entamoeba histolytica, Felis catus, Homo sapiens, Korat cats, Lactobacillus casei, Leishmania donovani, Mus musculus, Pisum sativum, Porphyromonas gingivalis, Pseudoalteromonas sp. S9, Rattus norvegicus, Serratia marcescens, Streptomyces plicatus, Streptomyces thermoviolaceus, Sus scrofa, Trichoderma harzianum, Vibrio furnissii, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vulnificus. The construct may be introduced transiently. The may also be introduced stably. The host cells may be selected from the group consisting of prokaryotic cells and eukaryotic cells. The method may further comprise the step of preparing membrane fractions of the cells. The step of determining the level of membrane-bound chitobiase activity may be selected from the group consisting of measuring the amount of a chemiluminescent product produced from a substrate, measuring the amount of a fluorescent product produced from a substrate, measuring the amount of light absorbed by a product produced from a substrate and measuring a decrease in the amount of a detectable substrate. The product may be p-nitrophenol. The test nucleic acid sequence may comprise a portion of genomic DNA.
The step of determining the level of membrane-bound chitobiase activity may comprise determining the level of membrane-bound chitobiase activity after exposing the host cells to a desired set of environmental conditions. The step of determining the level of membrane-bound chitobiase activity may comprise determining the level of membrane-bound chitobiase activity after contacting the host cells with a compound to be tested for its influence on the level of transcription from the regulatory element.
Another embodiment of the present invention is a method of detecting successful transformation, comprising the steps of introducing a nucleic acid encoding a membrane-bound form of chitobiase into host cells and detecting membrane-bound chitobiase expression in the host cells. The nucleic acid may encode a membrane-bound form of chitobiase obtained from an organism selected from the group consisting of Alteromonas sp. 0-7, Arabidopsis thaliana, Bacillus subtilis, Bombyx mori, Bos taurus, Caenorhabditis elegans, Candida albicans, Dictyostelium discoideum, Entamoeba histolytica, Felis catus, Homo sapiens, Korat cats, Lactobacillus casei, Leishmania donovani, Mus musculus, Pisum sativum, Porphyromonas gingivalis, Pseudoalteromonas sp. S9, Rattus norvegicus, Serratia marcescens, Streptomyces plicatus, Streptomyces thermoviolaceus, Sus scrofa, Trichoderma harzianum, Vibrio furnissii, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vulnificus. The nucleic acid may further comprise a xcex site-specific recombination sequence.
Another embodiment of the present invention is a method for monitoring the activity of a promoter comprising providing a construct comprising the promoter operably linked to a nucleic acid encoding a membrane-bound form of chitobiase, introducing the construct into host cells, and determining the level of membrane-bound chitobiase activity. The nucleic acid encoding a membrane-bound form of chitobiase may encode a membrane-bound form of chitobiase obtained from an organism selected from the group consisting of Alteromonas sp. 0-7, Arabidopsis thaliana, Bacillus subtilis, Bombyx mori, Bos taurus, Caenorhabditis elegans, Candida albicans, Dictyostelium discoideum, Entamoeba histolytica, Felis catus, Homo sapiens, Korat cats, Lactobacillus casei, Leishmania donovani, Mus musculus, Pisum sativum, Porphyromonas gingivalis, Pseudoalteromonas sp. S9, Rattus norvegicus, Serratia marcescens, Streptomyces plicatus, Streptomyces thermoviolaceus, Sus scrofa, Trichoderma harzianum, Vibrio furnissii, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vulnificus. The reporter gene construct may be introduced transiently. The reporter gene construct may be introduced stably. The reporter gene may be incorporated into the genome of the host cells. The host cells may be selected from the group consisting of prokaryotic cells and eukaryotic cells. The method may further comprise the step of preparing membrane fractions of the host cells. The step of determining the level of membrane-bound chitobiase activity may be selected from the group consisting of measuring the amount of a chemiluminescent product produced from a substrate, determining the level of chitobiase activity comprises measuring the amount of a fluorescent product produced from a substrate, measuring the amount of light absorbed by a product produced from a substrate and measuring a decrease in the amount of a detectable substrate. The product may be p-nitrophenol. The step of determining the level of membrane-bound chitobiase activity may comprise determining the level of membrane-bound chitobiase activity after exposing the host cells to a desired set of environmental conditions. The step of determining the level of membrane-bound chitobiase activity may comprise determining the level of membrane-bound chitobiase activity after contacting the host cells with a compound to be tested for its influence on the level of transcription from the regulatory element. The compound may comprise a compound to be tested for activity as a drug.
Another embodiment of the present invention is a method for determining whether a test protein is associated with the outer membrane, comprising the steps of: fractionating a cell population and assaying the fractions for membrane-bound chitobiase activity and test protein activity, wherein if the test protein and membrane-bound chitobiase are found in the same fraction, the test protein is a membrane protein. The test protein may be an antibiotic target.
Another embodiment of the present invention is a method of determining whether a test compound inhibits cellular proliferation comprising contacting a first population of cells expressing an ectoenzyme or a secreted enzyme with the test compound and comparing the activity of the ectoenzyme or the secreted enzyme in the first population of cells with the activity of the ectoenzyme or the secreted enzyme in a second population of cells expressing the ectoenzyme or the secreted enzyme, wherein the second population of cells was not contacted with the test compound and wherein if the level of activity of the ectoenzyme or the secreted enzyme in the first population of cells is significantly less than the level of activity of the ectoenzyme or the secreted enzyme in the second population of cells, then the test compound inhibits cellular proliferation. The ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase, P. aeruginosa FpvA ferric pyoverdin receptor, E. coli OmpP endopeptidase, outer membrane phospholipase A, Bacteriodes thetaiotamicron susG starch utilization protein, Haemophilus influenzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N-acetylglucosaminidase, S. pneumoniae neuraminidase, Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. The ectoenzyme or secreted enzyme may comprise a membrane-bound form of chitobiase.
The ectoenzyme secreted enzyme may be endogenous. The method may further comprise introducing a gene encoding the ectoenzyme or secreted enzyme into the cells prior to comparing the activity of the ectoenzyme or secreted enzyme in the first population of cells with the activity of the ectoenzyme or secreted enzyme in a second population of cells.
Another embodiment of the present invention is a method for identifying a compound which inhibits cellular proliferation comprising contacting a first population of cells expressing an ectoenzyme or secreted enzyme with the compound wherein the first population of cells has been sensitized by reducing the level or activity of a gene product required for proliferation and determining whether the compound inhibits cellular proliferation by detecting the activity of the ectoenzyme or secreted enzyme. The method may further comprise contacting a second population of cells expressing an ectoenzyme or secreted enzyme with the compound wherein the second population of cells has not been sensitized and comparing the activity of the ectoenzyme or secreted enzyme in the first population of cells with the activity of the ectoenzyme or secreted enzyme in the second population of cells, wherein the compound inhibits cellular proliferation if the level of activity of the ectoenzyme or secreted enzyme in the first population of cells is significantly less than the level of activity of the ectoenzyme or secreted enzyme in the second population of cells. The ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase, P. aeruginosa FpvA ferric pyoverdin receptor, E. coli OmpP endopeptidase, outer membrane phospholipase A, Bacteriodes thetaiotamicron susG starch utilization protein, Haemophilus influenzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N-acetylglucosaminidase, S. pneumoniae neuraminidase, Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. The ectoenzyme or secreted enzyme may comprise a membrane-bound form of chitobiase. The ectoenzyme or secreted enzyme may be endogenous.
Another embodiment of the present invention is a compound identified using the method of the preceding paragraph.
Another embodiment of the present invention is a method for identifying a compound which reduces the activity or level of a gene product required for proliferation of a microorganism wherein the activity or expression of the gene product is inhibited by an antisense nucleic acid, the method comprising the steps of (a) expressing a sublethal level of an antisense nucleic acid complementary to a nucleic acid encoding the gene product in a first population of cells expressing an ectoenzyme or secreted enzyme to reduce the activity or amount of the gene product in the cells, thereby producing sensitized cells (b) contacting the sensitized cells with a compound and (c) determining whether the compound alters cellular proliferation by measuring the level of activity of the ectoenzyme or secreted enzyme. The method may further comprise the steps of (d) contacting a second population of cells expressing an ectoenzyme or secreted enzyme with the compound and (e) comparing the activity of the ectoenzyme or secreted enzyme in the first population of cells with the activity of the ectoenzyme or secreted enzyme in the second population of cells, wherein the compound inhibits cellular proliferation if the level or activity of the ectoenzyme or secreted enzyme in the first population of cells is significantly less than the level or activity of the ectoenzyme or secreted enzyme in the second population of cells. The ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase, P. aeruginosa FpvA ferric pyoverdin receptor, E. coli OmpP endopeptidase, outer membrane phospholipase A, Bacteriodes thetaiotamicron susG starch utilization protein, Haemophilus influenzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N-acetylglucosaminidase, S. pneumoniae neuraminidase, Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. The ectoenzyme or secreted enzyme may be a membrane-bound form of chitobiase. The ectoenzyme or secreted enzyme amy be endogenous. The sensitized cell may contain an introduced gene encoding the ectoenzyme or secreted enzyme. The first population of cells may be from an organism selected from the group consisting of Staphylococcus aureus, Aspergillus fumigatus, Bacillus anthracis, Campylobacter jejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, E. coli, Enterobacter cloacae, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Salmonella cholerasuis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Staphylococcus epidermidis, Streptococcus pneumoniae, Treponema pallidunm, and Yersinia pestis or any species falling within the genera of any of the above species. The antisense nucleic acid may be transcribed from an inducible promoter. The method may further comprise the step of contacting the first population of cells with a concentration of inducer which induces the antisense nucleic acid to a sublethal level. The gene product may be a polypeptide. The gene product may be an RNA.
Another embodiment of the present invention is a compound identified using the method of the preceding paragraph.
Another embodiment of the present invention is a method for screening a test compound for activity against a gene or gene product that is essential for microbial proliferation, comprising providing a cell containing a gene encoding a gene product that is essential for microbial proliferation, wherein the cell further produces an ectoenzyme or secreted enzyme sensitizing the cell by reducing the activity or level of expression of the gene product contacting the sensitized cell with a test compound and determining whether the test compound alters cellular proliferation by mesuring the level of ectoenzyme or secreted enzyme activity. The sensitizing step may comprise contacting the cell with an antisense polynucleotide that inhibits production of the gene product. The ectoenzyme or secreted enzyme activity may be detected by detecting the action of the ectoenzyme or secreted enzyme on a substrate. The ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase, P. aeruginosa FpvA ferric pyoverdin receptor, E. coli OmpP endopeptidase, outer membrane phospholipase A, Bacteriodes thetaiotamicron susG starch utilization protein, Haemophilus influenzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N-acetylglucosaminidase, S. pneumoniae neuraminidase, Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. The ectoenzyme or secreted enzyme may be a membrane-bound form of chitobiase. The sensitizing step may comprise contacting the cell with an agent which reduces the activity or level of a gene product required for proliferation or growth of a microorganism. The agent may be a peptide or polypeptide. The cell may contain a mutation which reduces the activity or level of the gene product required for proliferation of the cell. The ectoenzyme or secreted enzyme may be endogenous.
Another embodiment of the present invention is a compound identified using the method of the preceding paragraph.
Another embodiment of the present invention is a method for identifying the biological pathway in which a proliferation-required gene or its gene product lies, wherein the gene or gene product comprises a gene or gene product whose activity or expression is inhibited by an antisense nucleic acid, the method comprising (a) expressing a sublethal level of an antisense nucleic acid which inhibits the activity or expression of the proliferation-required gene or gene product in a first population of cells expressing an ectoenzyme or secreted enzyme (b) contacting the first population of cells with a compound known to inhibit growth or proliferation of a microorganism, wherein the biological pathway on which the compound acts is known and (c) determining whether the compound alters cellular proliferation by measuring the level of activity of the ectoenzyme or secreted enzyme. The method may further comprise (d) contacting a second population of cells expressing an ectoenzyme or secreted enzyme with the compound and (e) determining whether the first population of cells has a significantly greater sensitivity to the compound than the second population of cells by comparing the activity of the ectoenzyme or secreted enzyme expressed by the first and second population of cells. The ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Bacteriodes thetaiotamicron susG starch utilization protein, Haemophilus influMoraxella (Branhamella) Catarrhalis BRO beta-lactamase, P. aeruginosa FpvA ferric pyoverdin receptor, E. coli OmpP endopeptidase, outer membrane phospholipase A, enzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N-acetylglucosaminidase, S. pneumoniae neuraminidase, Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. The ectoenzyme or secreted enzyme may be a membrane-bound form of chitobiase. The ectoenzyme or secreted enzyme may be endogenous.
Another embodiment of the present invention is a method for determining the biological pathway on which a test compound acts comprising (a) expressing a sublethal level of an antisense nucleic acid complementary to a proliferation-required nucleic acid in a first population of cells expressing an ectoenzyme or secreted enzyme, wherein the activity or expression of the proliferation-required nucleic acid is inhibited by the antisense nucleic acid and wherein the biological pathway in which the proliferation-required nucleic acid or a protein encoded by the proliferation-required polypeptide lies is known (b) contacting the first population of cells with the test compound and (c) determining whether the compound alters cellular proliferation by measuring the level of activity of the ectoenzyme or secreted enzyme. The method may further comprise (d) contacting a second population of cells with the test compound and (e) determining whether the first population of cells has a significantly greater sensitivity to the test compound that the second population of cells by comparing the activity of the ectoenzyme or secreted enzyme expressed by the cell populations. The ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase, P. aeruginosa FpvA ferric pyoverdin receptor, E. coli OmpP endopeptidase, outer membrane phospholipase A, Bacteriodes thetaiotamicron susG starch utilization protein, Haemophilus influenzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N-acetylglucosaminidase, S. pneumoniae neuraminidase, Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. The ectoenzyme or secreted enzyme may be a membrane-bound form of chitobiase. The ectoenzyme or secreted enzyme may be endogenous. The method may further comprise (f) expressing a sublethal level of a second antisense nucleic acid complementary to a second proliferation-required nucleic acid in a third population of cells, wherein the second proliferation-required nucleic acid is in a different biological pathway than the proliferation-required nucleic acid in step (a) and (g) determining whether the third cell does not have a significantly greater sensitivity to the test compound than a cell which does not express the sublethal level of the second antisense nucleic acid, wherein the test compound is specific for the biological pathway against which the antisense nucleic acid of step (a) acts if the third cell does not have significantly greater sensitivity to the test compound.
Another embodiment of the present invention is a method for manufacturing an antibiotic comprising the steps of screening one or more candidate compounds to identify a compound that reduces the activity or level of a gene product required for proliferation, wherein the effect of the compound on proliferation is determined by measuring the activity of an ectoenzyme or secreted enzyme expressed by the cell and manufacturing the compound so identified. The ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase, P. aeruginosa FpvA ferric pyoverdin receptor, E. coli OmpP endopeptidase, outer membrane phospholipase A, Bacteriodes thetaiotamicron susG starch utilization protein, Haemophilus influenzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA. S. pneumoniae beta-N-acetylglucosaminidase, S. pneumoniae neuraminidase, Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. The ectoenzyme or secreted enzyme may be a membrane-bound form of chitobiase. The gene product may comprise a gene product whose activity or expression is inhibited by an antisense nucleic acid. The ectoenzyme or secreted enzyme may be endogenous.
Another embodiment of the present invention is a method for identifying nucleic acids which inhibit cellular proliferation, comprising the steps of transcribing a first level of a nucleic acid in a first population of cells expressing a gene encoding an ectoenzyme or secreted enzyme and comparing the activity of the ectoenzyme or secreted enzyme in the first population of cells to the activity of the ectoenzyme or secreted enzyme in a second population of cells expressing the ectoenzyme or secreted enzyme, wherein the second population of cells transcribes the nucleic acid at a lower level than the first population of cells, or does not transcribe the nucleic acid, wherein the nucleic acid inhibits proliferation if the activity of the ectoenzyme or secreted enzyme is significantly less in the first population of cells than in the second population of cells. The nucleic acid may be a random genomic fragment. The nucleic acid may be an antisense nucleic acid. The nucleic acid may be a sense nucleic acid which encodes a peptide or polypeptide. The peptide or polypeptide may comprise a peptide or polypeptide that is normally expressed in the cell. The nucleic acid may encode an RNA comprising an RNA that is normally expressed inside the cell. The ectoenzyme or secreted enzyme may be selected from the group consisting of Pseudomonas aeruginosa metalloproteinase, Moraxella (Branhamella) Catarrhalis BRO beta-lactamase, P. aeruginosa FpvA ferric pyoverdin receptor, E. coli OmpP endopeptidase, outer membrane phospholipase A, Bacteriodes thetaiotamicron susG starch utilization protein, Haemophilus influenzae phosphomonoesterase, streptococcal protein Sir, streptococcal C5a peptidase, Lactococcus lactis serine protease NisP, proteinase PrtB, proteinase PrtH, proteinase PrtP, proteinase ScpA, S. pneumoniae beta-N-acetylglucosaminidase, S. pneumoniae neuraminidase, Streptococcus sobrinus dextranase, Streptococcus suis muramidase, Streptococcus mutans exo-beta-D-fructosidase, Staphylococcus aureus murine hydrolase, staphylococcal lipases, lysostaphin, endo-beta-N-acetylglucosaminidase, sulfhydryl protease, staphylococcal esterase, S. aureus nuclease, S. aureus fatty acid modifying enzyme, chitinase, S. aureus autolysin, hemolysin, DNase, coagulase, protein A, staphylokinase and enterotoxin. The ectoenzyme or secreted enzyme may bea membrane-bound form of chitobiase. The ectoenzyme or secreted enzyme may be endogenous. The nucleic acid may be transcribed from an inducible promoter. The transcribed nucleic acid may be a recombinant nucleic acid that has been introduced into the first and second populations of cells.
As used herein, the term xe2x80x9cproliferationxe2x80x9d means an increase in the number of cells with time. By xe2x80x9cinhibition of proliferationxe2x80x9d is meant that growth, replication or viability of the microorganism is reduced or eliminated.